Induction of proliferation and cytokine production in human T lymphocytes by lipopolysaccharide (LPS).

Lipopolysaccharide (LPS), also known as endotoxin, is a compound of the cell wall of Gram-negative bacteria, which has been demonstrated to induce inflammatory reactions in vitro as well as in vivo, including lethal shock. A great number of different cells have been documented to be reactive to LPS, e.g. monocytes/macrophages, vascular cells, polymorphonuclear cells, and even B lymphocytes. We have now established that T lymphocytes could also contribute to an inflammatory reaction to LPS. LPS is a potent inducer of human T-lymphocyte proliferation and cytokine production. The activation of T lymphocytes by LPS requires direct cell-to-cell contact with viable accessory monocytes. This interaction was found to be MHC-unrestricted, but strongly dependent on costimulatory signals provided by B7/CD28 interactions. The frequency of responding T lymphocytes is less than 1:1000. A very exciting finding was that not only monocytes, but also CD34+ hematopoietic stem cells, which circulate in peripheral blood in very low frequency, exert essential accessory cell activity during stimulation of T lymphocytes by LPS. In contrast, the response of T lymphocytes to conventional recall antigens is not controlled by blood stem cells. These conclusions are based on the observation that depletion of CD34-positive blood stem cells resulted in a complete loss of LPS-induced T-lymphocyte stimulation. Addition of CD34-enriched blood stem cells led to a recovery of reactivity of T lymphocyte to LPS. The characteristics of T-lymphocyte activation indicate that LPS is neither active as a mitogen, or as a superantigen, or as a classical antigen, but may activate T lymphocyte through a new, so far undescribed, mechanism. Furthermore, the involvement of hematopoietic blood stem cells in the activation of T lymphocytes by LPS demonstrates a role of these cells in inflammatory and immunological events.

In vitro generation of bacillus Calmette-Guérin-activated killer cells.

Tumor regression induced in cancer patients by local instillation of bacillus Calmette-Guérin (BCG) into the bladder is considered to be mediated by cellular immune and inflammatory reactions. In an attempt to elucidate which of these effects are relevant to tumoricidal activity, an in vitro system was employed in which the immunostimulatory effects of BCG could be studied. This report describes the induction of BCG-activated killer (BAK) cells, which effectively lyse bladder tumor cells. Human peripheral blood mononuclear cells (PBMC) were stimulated with viable and sonicated BCG (v-BCG and s-BCG, respectively) to generate BAK cells. Cytotoxicity of BAK cells was comparable with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interferon (IFN)-gamma but did not reach the level of interleukin-2 (IL-2)-generated LAK cells. Induction of BAK cells was possible only with v-BCG and not with s-BCG. By depletion and enrichment of defined cell populations, the cytotoxic potential of BAK cells could be attributed to a population of CD8(+) and CD56(+) double-positive lymphocytes. Macrophages and CD4(+) cells were required for the induction of killing activity but had no such activity by themselves. Furthermore, the presence of IFN-gamma and IL-2 in the supernatants harvested during the generation of BAK cells was demonstrated. Monoclonal antibodies neutralizing these cytokines abolished BCG-mediated cytotoxicity. From these results, it is concluded that the known beneficial effect of local instillation of BCG on maintenance of the relapse-free state in superficial bladder cancer may be due to local generation of BAK cells.

CD34(+) hematopoietic stem cells exert accessory function in lipopolysaccharide-induced T cell stimulation and CD80 expression on monocytes.

CD34(+) hematopoietic stem cells, which circulate in peripheral blood with very low frequency, exert essential accessory function during lipopolysaccharide (LPS)-induced human T lymphocyte activation, resulting in interferon gamma production and proliferation. In contrast, stimulation of T cells by "conventional" recall antigens is not controlled by blood stem cells. These conclusions are based on the observation that depletion of CD34(+) blood stem cells results in a loss of LPS-induced T cell stimulation as well as reduced expression of CD80 antigen on monocytes. The addition of CD34-enriched blood stem cells resulted in a recovery of reactivity of T cells and monocytes to LPS. Blood stem cells could be replaced by the hematopoietic stem cell line KG-1a. These findings may be of relevance for high risk patients treated with stem cells or stem cell recruiting compounds and for patients suffering from endotoxin-mediated diseases.

CD26 (dipeptidyl peptidase i.v.) on human T lymphocytes does not mediate adhesion of these cells to endothelial cells or fibroblasts.

We have examined the role of CD26 (dipeptidyl peptidase IV) in the adhesion of resting and activated T lymphocytes to endothelial cells and fibroblasts. For this purpose, we ran a short-time adhesion assay under different strategies: Adhesion of T lymphocytes was determined in the presence of different anti-CD26 monoclonal antibodies, or in the presence of synthetic inhibitors of the enzymatic function of CD26. In addition, the expression of CD26 on T lymphocytes, which were adherent to endothelial cells or fibroblasts, was performed by flow cytometric analysis. We found that the anti-CD26 monoclonal antibodies tested here were not able to inhibit T cell adhesion to monolayers of endothelial cells or fibroblasts. Secondly, synthetic inhibitors of the enzymatic function of CD26 had no effect on the adhesion of T lymphocytes to endothelial cells or fibroblasts. Furthermore, CD26-positive T cells were not accumulated in the adherent population. These results suggest that CD26 on T lymphocytes plays no role in T cell adhesion to endothelial cells or fibroblasts.

Staining pattern of seven monoclonal anti-CD26 antibodies in leprosy: implications for the use of CD26 as a surrogate marker of a human Th1-like reaction.

In a previous study using the monoclonal anti-CD26 antibody MIB-DS2/7 in leprosy and other granulomatous diseases, it was shown that CD26 may be a candidate for use as an operational marker of a human Th1-like reaction. In this follow-up study, we compared seven different monoclonal anti-CD26 antibodies with respect to their staining pattern in lepromatous and tuberculoid leprosy tissues. Three distinct staining patterns became apparent in this anti-CD26 antibody panel: staining of T-lymphocytes and of connective tissue; staining of T-lymphocytes, connective tissue and macrophages; and almost no staining of T-lymphocytes but staining of connective tissue and macrophages. The two antibodies assigned to the first staining pattern, including MIB-DS2/7, were found to be most suitable for the operational discrimination between Th1-like and Th2-like reactions in leprosy. The antibodies assigned to staining patterns 2 and 3 did not allow this discrimination. Although all seven monoclonal antibodies investigated were specific for CD26, only two were found to be useful in identifying a Th1-like immune reaction in human tissue.

Stimulation of human T lymphocytes by LPS is MHC unrestricted, but strongly dependent on B7 interactions.

Recently, we have shown that LPS is a potent inducer of human T cell proliferation and lymphokine production. However, the activation of T cells by LPS has been demonstrated to be monocyte dependent and to require direct cell-to-cell contact. Here, we investigated the role of monocytes as accessory cells and the requirement for costimulatory signals in more detail. We found that the accessory cell activity of monocytes during LPS-induced T cell proliferation is characterized by the following features: LPS-primed monocytes are competent stimulators of T cell proliferation; interaction of LPS with monocytes during the priming step is dependent on CD14 and is sensitive to ammonia; monocyte/T cell interactions are not MHC restricted but are strongly dependent on interactions of CD28 and/or CTLA-4 on T cells and their ligands CD80 and/or CD86 on monocytes. CD80 seems to be crucial for the activation of T cells by monocytes, since monocytes expressing CD86 but not CD80 after LPS stimulation were unable to stimulate T cells; IL-12, at least as a costimulatory factor, but not IL-15, is important in LPS-induced T cell proliferation. Taken together, our results indicate that LPS acts neither as a mitogen, nor as a superantigen, nor as an Ag. The activation of human T cells by LPS requires the help of accessory functions by primed monocytes and is MHC unrestricted but needs costimulatory signals via CD28 and/or CTLA-4.

Lipopolysaccharide and peptidoglycan: CD14-dependent bacterial inducers of inflammation.

Surface structures of bacteria contribute to the microbial pathogenic potential and are capable of causing local and generalized inflammatory reactions. Among these factors, endotoxin and peptidoglycan are of particular medical importance. Both toxic bacterial polymers are now recognized to interact with the same cellular receptor, the CD14 molecule, which is expressed on different types of immune cells, in particular, monocytes/macrophages. The interaction between these bacterial activators and CD14 leads to the production of endogenous mediators such as tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-6, which are ultimately responsible for phlogistic responses. The fact that CD14 recognizes not only endotoxin and peptidoglycan but also other glycosyl-based microbial polymers suggests that this cellular surface molecule represents a lectin.

Comparative study of CD26 as a Th1-like and CD30 as a potential Th2-like operational marker in leprosy.

In the last years we have been able to establish CD26 as an operational marker for a human Th1-like reaction in various granulomatous diseases. Recently, CD30 was described as a marker for a Th2-type reaction, where CD30 is preferentially expressed and its soluble form released by human T cell clones producing Th2-type cytokines. To evaluate the possibility of CD30 as an eventual operational marker for a human Th2-like reaction in vivo, we performed immunohistological stainings on frozen sections of skin biopsies from patients with lepromatous and tuberculoid leprosy. A maximum of three to four CD30-positive cells was found per section, and there was no difference in the accumulation of CD30-positive cells between the tuberculoid and the lepromatous form of leprosy. With respect to CD26-positive cells, a high number was found in tuberculoid leprosy in contrast to a greatly reduced expression of CD26 in lepromatous leprosy. We conclude that, while CD26 was confirmed as an operational marker for a Th1-like reaction in leprosy, CD30 does not represent an operational Th2 marker in this disease.

Antibody-induced modulation of CD26 surface expression.

The ability of different anti-CD26 monoclonal antibodies to modulate the expression of CD26 on human T lymphocytes was investigated. By means of a new non-radioactive method using fluorescein isothiocyanate (FITC)-labelled and unlabelled anti-CD26 monoclonal antibodies and flow cytometry, we measured the internalization and re-expression of CD26 on freshly isolated resting human T lymphocytes. The modulation of CD26 surface expression takes place in primarily CD26+ as well as in CD26- T lymphocytes, indicating the presence of an intracellular CD26 pool. In fact, with two different anti-CD26 monoclonal antibodies (Ta1 and M5) intracellular CD26 was detected out of which newly expressed CD26 might have originated. This intracellular CD26 pool appears to be maintained by continuous translation of CD26 mRNA.

The induction of bacillus-Calmette-Guérin-activated killer cells requires the presence of monocytes and T-helper type-1 cells.

Previously we have described the induction of MHC-unrestricted killer cells against bladder tumour cells by bacillus Calmette-Guérin (BCG), termed BCG-activated killer (BAK) cells. In the present paper we deal with the accessory-cell requirement for the activation of BAK cells. We show that monocytes are required for activating BAK cells, since no cytotoxicity can be induced in the absence of monocytes. Therefore, these phagocytes may represent the first step during the activation cascade of BAK cells. Furthermore, the presence of CD4+ T cells was essential for generating BAK cells: depleting peripheral blood mononuclear cells of CD4+ cells prior to stimulation with BCG abolished the cytotoxicity against bladder tumour cells. Experiments with monoclonal antibodies (mAb) neutralizing the activity of either interleukin-2 (IL-2) or interferon gamma (IFN gamma) underlined the importance of these cytokines: both mAb blocked the induction of BAK cells. Since both cytokines are related to the so-called Th1 pattern of T cells, we consider the second step of the generation of BAK cells as follows: monocytes presenting antigens of BCG trigger Th1-like cells in a preferred manner. These Th1-like T cells secrete IL-2 and IFN gamma and, thus, activate the BAK effector cells. Since CD4+ cells are dominant in the cells infiltrating the bladder wall after intravesical instillation of BCG in vivo, we postulate an important role for the Th1 subpopulation. We further postulate that the occurrence of macrophages in this infiltrate seems to be significant in the maintenance of the relapse-free state of the patient.

Prevention of venous thromboembolism in North America: results of a survey among general surgeons.

PURPOSE: The purpose of this study was to analyze current attitudes toward the prevention of postoperative venous thromboembolism among North American general surgeons. METHODS: A survey regarding awareness of the problem of venous thromboembolism and preferred modalities of prophylaxis was sent to 3500 randomly selected Fellows of the American College of Surgeons. RESULTS: A total of 1018 (29.1%) surveys was returned. Most of the responding surgeons consider venous thromboembolism a serious health problem. Ninety percent of the surgeons use prophylaxis against venous thromboembolism routinely. The most frequently used modalities are intermittent pneumatic compression, low-dose heparin, and elastic stocking. A combination of physical and pharmacologic methods is used by one fourth of respondents, and only 50% start pharmacologic prophylaxis before the surgical procedure. The thrombosis risk factors that are most frequently considered by surgeons when deciding about using prophylaxis are history of venous thromboembolism, immobility, and length of operation. CONCLUSIONS: North American surgeons who responded to this survey are well aware of the problem of venous thromboembolism and their approach to prevention has been significantly modified in the last 10 years. Compared with similar European surveys this survey reveals a higher implementations of physical methods such as intermittent pneumatic compression and elastic stockings. Because of the limited response rate and possibility of sampling bias, these findings should be interpreted with caution.

Endotoxin and lipid A stimulate proliferation of human T cells in the presence of autologous monocytes.

In this paper we describe a new activity of LPS and partial structures: the induction of DNA synthesis and lymphokine production of human T lymphocytes. The LPS-induced T cell proliferation is dose dependent and requires 100 to 10,000 ng/ml of LPS or synthetic lipid A (compound 506) for optimal stimulation. In contrast, the synthetic lipid A precursor Ia (compound 406) is not active but rather antagonizes LPS-induced proliferation. The proliferation is accompanied by the expression of mRNA for the Th1 cell-derived lymphokines IFN-gamma and IL-2, but not for the Th2 lymphokines IL-4, IL-5, or IL-10. Highly enriched T lymphocyte preparations with less than 0.1% monocytes are not stimulated by LPS, showing that monocytes are required for T cell proliferation. Reconstitution experiments show that only monocytes, but not B lymphocytes, are able to support induction of DNA synthesis. Separating LPS-stimulated monocytes from T lymphocytes by a membrane, permeable for cytokines but not for cells, abolishes T cell proliferation. Fixation of monocytes with paraformaldehyde also abrogates their accessory function for T cells. If the monocytes are preincubated for 2 h at 37 degrees C with LPS and then washed, they still are able to induce T cell proliferation in the absence of additional LPS. Our results indicate that human T cells respond in a monocyte-supported manner to LPS exposure by proliferation and lymphokine production. We hypothesize that this reactivity of T lymphocytes to LPS may be of clinical relevance.

On the mode of action of intravesical bacillus Calmette-Guérin: in vitro characterization of BCG-activated killer cells.

Previously we had shown that, upon activation with viable bacillus Calmette-Guérin (BCG), peripheral blood mononuclear cells (PBMNC) could be rendered cytotoxic against otherwise insensitive natural killer (NK)-resistant bladder cancer cell lines. This phenomenon had been termed the BCG-activated killer (BAK) cell phenomenon. By means of depletion and enrichment procedures of mononuclear cell subpopulations derived from BCG-activated PBMNC we further characterized the cytolytic BAK effector cells functionally in an in vitro cytotoxicity assay against the bladder carcinoma cell line BT-A and phenotypically in their pathway of activation. Neither macrophages nor CD4+ T-helper/inducer cells exerted cytotoxic BAK activity. This cytotoxicity was restricted to the CD8+CD56+ subpopulation of T-cytotoxic/NK cells. Furthermore, activation of BAK cells via interferon gamma (IFN-gamma) was evidenced by the complete inhibition of BAK cell generation with an IFN-gamma antibody.

Dipeptidyl peptidase IV (CD26) on human lymphocytes. Synthetic inhibitors of and antibodies against dipeptidyl peptidase IV suppress the proliferation of pokeweed mitogen-stimulated peripheral blood mononuclear cells, and IL-2 and IL-6 production.

In the present report, we describe that synthetic inhibitors of and polyclonal and monoclonal antibodies against the membrane ectoenzyme dipeptidyl peptidase IV (DP IV, CD26) inhibit the production of IL-2 and IL-6 and, concomitantly, DNA synthesis of pokeweed mitogen-stimulated peripheral blood mononuclear cells (PBMC). The release of IL-1 and TNF-alpha, was not influenced under these conditions. The data support the hypothesis that DP IV, possibly in conjunction with other peptidase, is involved in the regulation of activation and proliferation of T lymphocytes.

Induction of bacillus-Calmette-Guérin-activated killer cells from human peripheral blood mononuclear cells against human bladder carcinoma cell lines in vitro.

Cytotoxicity against two human bladder carcinoma cell lines (BT-A and BT-B) was investigated using human peripheral blood mononuclear cells (PBMC) stimulated with viable bacillus Calmette-Guérin (BCG) or sonicated BCG (s-BCG). We applied a cytotoxicity assay based on radioactive labelling of tumour cells by incorporation of L[3H]methionine. The results were compared with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interleukin-2 (IL-2) and interferon gamma (IFN gamma). BCG-stimulated PBMC showed a cytotoxic potential against BT-A and BT-B comparable to that of IFN gamma-generated LAK cells, but this did not reach the level of IL-2-generated LAK cells. We termed these cytotoxic effectors BCG-activated killer (BAK) cells. In contrast to their cytotoxicity against bladder tumour cells, BAK cells did not differ from unstimulated PBMC in the killing of K562 cells. Only viable but not sonicated BCG was able to induce cytotoxicity against BT-A and BT-B. We could demonstrate the presence of the cytokines IFN gamma, IL-2, tumour necrosis factor alpha (TNF alpha) and TNF beta in the supernatants harvested during the generation of BAK cells. Monoclonal antibodies neutralizing IFN gamma were able to inhibit BCG-mediated cytotoxicity, giving evidence of the involvement of IFN gamma in the induction of BAK cells. Furthermore, we performed experiments to investigate the cytotoxic potential of distinct cell populations. The cells effective in BCG-activated killing of bladder tumour cells could be localized within the CD8+/CD56+ lymphocyte subset. CD4+ cells and macrophages did not exhibit cytolytic activity. Our findings imply that the activation by BCG of CD8+/CD56+ killer cells might be an important antitumoral mechanism during BCG therapy against superficial urothelial bladder cancer.

Anti-CD26 monoclonal antibodies can reversibly arrest human T lymphocytes in the late G1 phase of the cell cycle.

Three different anti-CD26 monoclonal antibodies (mAbs) are described, which specifically inhibited proliferation of human T lymphocytes after stimulation with PHA, tetanus toxoid or soluble anti-CD3 mAb. Anti-CD26 mAbs induced in T cells a dose-dependent shift of the maximum of DNA synthesis, which was due to a transitory arrest of cells in the cell cycle. This cell cycle arrest was found to occur in the late phase of G1 since the expression of the T cell activation markers CD25-, CD71-, and HLA-DR-positive cells was the same in anti-CD26 mAb-containing and control cultures. Propidium iodide staining further confirmed the assumption that the arrest occurs in the first round of the cell cycle before S phase cells were detectable. Because the cells were arrested before consuming IL-2 in the S phase, we observed in accumulation of IL-2 in anti-CD26 mAb-containing cultures, whereas IFN-gamma production by PHA-activated T lymphocytes was reduced. These data indicate that CD26 is involved in the processes of T cell activation and proliferation.